In hearts from three different transgenic mouse models (Na-Ca overexperssor, beta-2-adrenergic overexpressor, phospholamban KO), which all would be expect to have an increase in sarcoplasmic reticulum (SR) calcium, we found that males but not females have poorer recovery of function following stress. The functional recovery of hearts from bilateral ovariectomized female transgenic mice was significantly worse than that of non-ovariectomized female transgenics and similar to that of male transgenic mice, suggesting a role for estrogen. Although we do not typically observe male/female differences in response to injury in wild type control mice, such differences can be observed if we raise the extracellular calcium or treat hearts with isoproterenol, conditions which would elevate cell and SR calcium. We directly measured sarcoplasmic reticulum calcium in male and female hearts treated with isoproterenol using a 19F NMR sensitive high Kd calcium indicator, and preliminary data suggest that isoproterenol results in a smaller increase n SR Ca2+ in females than in males. The protection in females appears to be related to an increase in eNOS activity, likely due to increased phosphorylation by PKB. Interestingly this pathway is also involved in the protection afforded by PC. Increased injury in males can be ameliorated with nitric oxide synthase inhibitors. We also find an increase in eNOS and an increase in NO production in females. To further investigate the effect of NO on SR Ca2+ we treated perfused hearts with the NO donor, SNAP, and measured SR Ca2+ with the 19F NMR indicator. We found that SNAP significantly reduced SR Ca2+. In addition, western blot analysis showed that SNAP resulted in nitrosylation of tyrosine in SERCA. Taken together these data suggest that altered NO signaling leading to modification of SERCA and SR Ca2+ are important mediators of gender differences in response to injury. The potential role of the phosphatidylinositol 3-kinase (PI3K) pathway in reduced isoproterenol-induced ischemia/reperfusion injury was investigated. Langerdorff perfused male and female mouse hearts were subjected to a 20 min treatment period, 20 min of global ischemia and 2 hr of reperfusion while monitoring pH and high energy phosphates with nuclear magnetic resonance and left ventricular developed pressure (LVDP) via a fluid filled balloon in the left ventricle connected to a pressure transducer. During the treatment period hearts were perfused in the presence or absence of 100nM wortmannin (WM) for 20 min followed by 1 min perfusion with 10 nM isoproterenol (iso). In the absence of WM. females exhibited significantly better functional recovery compared with males (17.0+/-2.5% vs. 8.2+/-3.5% p<0.05)). To test whether this protection might be mediated through the PI3K pathway, female hearts were treated with PI3K inhibitor WM. Inhibition of PI3K blocked the protection seen in females, as functional recovery was significantly worse in the WM/ iso treated female hearts than in the females that did not receive WM treatment (7.0?2.9%; p<0.05), and not significantly different than males treated with WM. We also performed studies to determine whether the beta-estrogen receptor was involved in cardioprotection in females treated with isoproterenol. This study utilized female mice which were null for the beta-estrogen receptor (BERKO) and compared them to wild-type (WT) females. The hearts were subjected to 25 minutes of global ischemia, followed by a 2 hour reperfusion period. Ischemic damage was assessed by measuring recovery of function (percent of control; rate-pressure product) and infarct size. Infarct size was measured by perfusing the heart with 1% 2,3,5-triphenyltetrazolium chloride following the 2hr reflow period. Transverse slices through the ventricles were then quantitated for percent of necrosis. This study indicates that mice which are null for ?-estrogen receptors exhibit a significantly improved recovery of function compared to WT. Infarct size also tended to be smaller in hearts from BERKO mice. These data suggest that the absence of the beta-estrogen receptor is protective.